PROJECT ABSTRACT E-cigarette (e-cig) aerosol/vapor is a complex mixture of the original components of the e-liquid (propylene glycol (PG), vegetable glycerol (VG), nicotine, water, and flavoring additives), and of other constituents (such as aldehydes, metals, nanoparticles, and some unknown compounds) produced by chemical transformation of the original components exposed to the electrically heated metal wire in the presence of oxygen. Emerging research is beginning to challenge the ?relatively safe? perception of e-cigarettes. Recent studies suggest e-cig aerosol/vapor provokes an inflammatory response and oxidative stress (reminiscent of cigarette smoke where oxidative stress and inflammation are among the first clinically defined events associated with toxicity and disease due to cigarette smoking) however details of the underlying molecular mechanisms remain unclear. Furthermore the contributions that specific e-liquid constituents have in mediating them as well as the impact of oxidation products of e-liquid constituents generated by the e-cigarette heating process, are poorly understood. Focusing on oxidative stress and inflammation as intermediate measures of biological responses predictive of tissue dysfunction leading to disease initiation we will assess the hazard potential of e-liquid main components (PG, VG, nicotine, and flavorings). We hypothesize that individual components of e-liquid produce distinct signatures of early oxidative/nitrative damage in cells and tissue. Our objectives are to elucidate the signatures of reversible and irreversible oxidative modifications induced by e-cig aerosol and use them to evaluate the relative hazards of different e-liquid constituents in conjunction with computational fluid dynamic- physiologically-based pharmacokinetic (CFD/PBPK) models for comparative respiratory dosimetry. Most commonly used measures of cell redox state (i.e., GSH content) provide little insight into the types or sites of damage induced critical for a mechanistic understanding. Recent advances in quantitative redox proteomics at PNNL make it feasible to identify these modifications and determine their site-specific occupancies at a proteome-wide scale. The Research Grade E-cigarette (REC) device developed at Battelle provides a unique capability to generate and characterize aerosol/vapor from individual e-liquid components with or without the use of a heated coil, allowing effects of both the major e-liquid components as well as potential toxicants that result from (coil) heating of the e-liquid in the presence of oxygen to be identified and quantified. Using these novel enabling technologies, we will define what compounds or aerosol size fraction are most harmful and how they contribute to the effects of inhalation of e-cig aerosol and individual aerosolized e-liquid components on pulmonary response. We will accomplish our goal through the following aims: (1) Characterize aerosol/vapor of e-cigarette generated at moderate and high heating temperature (2) Molecularly characterize pulmonary response to e-cig aerosol/vapor and e-cig aerosol/vapor components exposure in mice (3) Predict regional (airway) deposition and site-specific tissue dose and translation to humans using CFD/PBPK models.